Thrombosis is caused by fibrin and platelet deposits that occlude blood vessels in various organs. The role of thrombosis in morbidity and mortality has been documented in many diseases, including, among others, deep vein thrombosis, pulmonary thrombo-embolism, myocardial infarction, ischemic stroke, anthrax and meningococcal sepsis, and heparin-induced thrombocytopenia. Macrovascular thrombosis can be prevented or successfully treated with anticoagulants, antiplatelet agents, and/or profibrinolytic agents. The antithrombotic therapy for microvascular thrombosis, however, presents a greater medical challenge. Pharmacological use of activated protein C (APC), a naturally circulating anticoagulant enzyme (Gruber et al. Blood 79: 2340-2348 (1992)) has been shown to reduce the mortality of severe sepsis (Bernard et al. New Eng. J. Med. 344: 699-709 (2001)). Clinical use of APC is now medically justifiable. However, manufacturing of injectable dosage forms of natural or recombinant APC for therapeutic use is expensive, especially in view of the large doses, such as, for example, administering 0.024 mg/kg/hour of recombinant human APC for the several days required for effective treatment.
The activation of naturally occurring physiologic systems leading to the production of endogenous therapeutic proteins can be as efficacious and more economical than administering the directly acting agent itself. For example, relatively inexpensive plasminogen activators, such as streptokinase, are valuable in the systemic treatment of thrombosis, while the directly acting enzyme, plasmin, is suitable for topical therapy only (Marder et al. Thromb. Haemost. 86: 739-745 (2001)). An affordable alternative modality is needed to make APC-therapy accessible to a broader patient population, including those who suffer from septic disseminated intravascular coagulation due to sepsis.
Low dose wild-type human thrombin (WT) is a relatively safe antithrombotic agent in baboons (Hanson et al. J. Clin. Invest. 92: 2003-2012 (1993)) that is capable of binding to thrombomodulin and generating endogenous APC. However, the low-grade fibrin formation and platelet activation that accompany the infusion of WT have potentially adverse side-effects. WT not complexed with thrombomodulin can cause potentially fatal disseminated intravascular coagulation (Gresele et al. J. Clin. Invest. 101: 667-676 (1998)). Thrombomodulin deficiency or poor microcirculation in a patient pose additional safety risks. The use of guanidinobenzoyl thrombin for protein C activation has addressed several of these problems of WT because acyl-thrombin yields active thrombin by delayed deacylation after binding to endothelial thrombomodulin (McBane et al. Thromb. Haemost. 74: 879-885 (1995)). Acyl-thrombin is effective with a wider safety margin than WT in a pig model of thrombosis. The acylation approach, however, reduces, but does not eliminate, the potentially disastrous consequences of an inadvertent overdose, when simultaneous deacylation of unbound acyl-thrombin would suddenly clot all circulating blood.
Activation of the circulating protein C pool by a suitable snake venom activator has also been shown to produce antithrombotic effects in an arterio-venous (AV) shunt model (Kogan et al. Thromb. Res. 70: 385-393 (1993)). There are several potential advantages to snake enzymes, such as high specificity, long half-life, and stability. Immunogenecity, however, does present a problem if used repeatedly. Also, these enzymes are not readily available. In search for newer, specific, and safe protein C activators, a number of thrombin mutations have been reported to compromise cleavage of fibrinogen more than the activation of protein C (Cantwell et al. J. Biol. Chem. 275:39827-39830 (2000); Wu et al. Proc. Natl. Acad. Sci. U.S.A. 88:6775-6779 (1991); Gibbs et al. Nature 378: 413-416 (1995); Arosie et al. Biochemistry 39: 8095-8101 (2000)).
What is still needed, however, is an antithrombotic thrombin with a substantially reduced procoagulant activity and a compromised platelet activation activity, but having an effective capability to activate protein C.
What is also needed, therefore, is a variant thrombin that is practically devoid of activity towards fibrinogen and the platelet receptor PAR-1, but retains a significant capability to activate protein C in the presence of thrombomodulin.
What is still further needed are methods of administering to a patient variant thrombins capable of activating protein C but not of inducing thrombus formation.
What are also needed are methods to determine the antithrombotic potential and the status of activated protein C in the blood of an animal or human that do not necessitate the use of expensive protein C activators or the use of a protein C activator capable of inducing thrombus formation.